The point mutation UCH-L1 C152A protects primary neurons against cyclopentenone prostaglandin-induced cytotoxicity: implications for post-ischemic neuronal injury.
The point mutation UCH-L1 C152A protects primary neurons against cyclopentenone prostaglandin-induced cytotoxicity: implications for post-ischemic neuronal injury.
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DOI：
10.1038/cddis.2015.323
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发表时间：
2015-11-05
影响因子：
9
通讯作者：
Graham SH
中科院分区：
文献类型：
--
作者：
Liu H;Li W;Rose ME;Hickey RW;Chen J;Uechi GT;Balasubramani M;Day BW;Patel KV;Graham SH
环戊烯酮前列腺素 (CyPG)，例如 15-脱氧-Δ12,14-前列腺素 J2 (15dPGJ2)，是具有多种生物效应的反应性前列腺素代谢物。 CyPG 在缺血性大脑中产生，会破坏泛素蛋白酶体系统 (UPS)。泛素 C 末端水解酶 L1 (UCH-L1) 是一种大脑特异性去泛素化酶，与神经退行性疾病有关。使用串联质谱 (MS) 分析，我们发现 UCH-L1 的 C152 位点被 CyPG 加合。 C152 突变为丙氨酸 (C152A) 可抑制 CyPG 修饰并在 15dPGJ2 处理后保留重组 UCH-L1 蛋白水解酶活性。采用细菌人工染色体（BAC）技术构建表达UCH-L1 C152A突变的敲入（KI）小鼠。通过蛋白质印迹法测定，KI 小鼠中 UCH-L1 的脑表达和分布与野生型 (WT) 相似。通过 WST-1 细胞活力测定以及 caspase-3 和聚 ADP 核糖聚合酶 (PARP) 裂解检测到，与 WT 小鼠的神经元相比，KI 小鼠的原代皮质神经元对 15dPGJ2 细胞毒性具有抵抗力。 15dPGJ2 处理后，这种保护作用伴随着 C152A KI 原代神经元中泛素化蛋白积累和聚集的显着减少以及 UCH-L1 聚集的减少。此外，与 WT 相比，KI 神经元中 15dPGJ2 诱导的轴突损伤也显着减弱。综上所述，这些研究表明UCH-L1功能在缺氧神经元死亡中很重要，并且UCH-L1的C152位点在缺氧/缺血性损伤后神经元存活中具有重要作用。
Cyclopentenone prostaglandins (CyPGs), such as 15-deoxy-Δ12,14-prostaglandin J2 (15dPGJ2), are reactive prostaglandin metabolites exerting a variety of biological effects. CyPGs are produced in ischemic brain and disrupt the ubiquitin-proteasome system (UPS). Ubiquitin-C-terminal hydrolase L1 (UCH-L1) is a brain-specific deubiquitinating enzyme that has been linked to neurodegenerative diseases. Using tandem mass spectrometry (MS) analyses, we found that the C152 site of UCH-L1 is adducted by CyPGs. Mutation of C152 to alanine (C152A) inhibited CyPG modification and conserved recombinant UCH-L1 protein hydrolase activity after 15dPGJ2 treatment. A knock-in (KI) mouse expressing the UCH-L1 C152A mutation was constructed with the bacterial artificial chromosome (BAC) technique. Brain expression and distribution of UCH-L1 in the KI mouse was similar to that of wild type (WT) as determined by western blotting. Primary cortical neurons derived from KI mice were resistant to 15dPGJ2 cytotoxicity compared with neurons from WT mice as detected by the WST-1 cell viability assay and caspase-3 and poly ADP ribose polymerase (PARP) cleavage. This protective effect was accompanied with significantly less ubiquitinated protein accumulation and aggregation as well as less UCH-L1 aggregation in C152A KI primary neurons after 15dPGJ2 treatment. Additionally, 15dPGJ2-induced axonal injury was also significantly attenuated in KI neurons as compared with WT. Taken together, these studies indicate that UCH-L1 function is important in hypoxic neuronal death, and the C152 site of UCH-L1 has a significant role in neuronal survival after hypoxic/ischemic injury.